This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Platelet- and plasma-derived factor Va are essential for thrombin generation at sites of vascular injury. Though plasma-derived factor Va is sufficient for normal hemostasis, it has been demonstrated that platelet-derived factor Va is functionally and physically distinct, and represents the physiologically-relevant cofactor molecule. Despite these differences, the platelet-derived procofactor, factor V, originates from plasma through endocytosis of plasma-derived factor V by platelet precursors, megakaryocytes, via a two receptor system consisting of a specific, unidentified factor V receptor and LDL receptor-related protein-1 (LRP-1). In this model, it is hypothesized that factor V binding to its specific receptor facilitates binding of another factor V molecule to LRP-1 suggesting that the two receptors are physcially-associated on the cell surface. Factor V endocytosis is subsequently mediated by LRP-1. This represents a novel role for LRP-1 in endocytosis of a protein physically and functionally modified, and not destined for lysosomal degradation. The goal of this project is to continue to characterize this two receptor system by identifying the specific, factor V receptor by mass spectrometry following co-immunoprecipitation with anti-LRP-1 antibodies. As early attempts to detect the ligand binding domain of LRP-1 by western blotting of megakaryocyte cell lysates were unsucessful due to its high molecular weight (550 kDa), protocols were developed to define appropriate western blotting conditions. Blotting specificity was confirmed using lysates from LRP-1 positive or negative cells. Following incubation of megakaryocytes with SBED-biotin-derivatized receptor associated protein, which antagonizes LRP-1 ligand binding, the biotin moeity was transferred to LRP-1 as confirmed by streptavidin and western blotting. However, attempts to precipitate biotinylated LRP-1 using streptavidin or anti-LRP-1 antibodies have been unsucessful. Current experiments are assessing the the use of SBED-biotin-derivatized factor V light chain to biotinylate and isolate the specific, factor V receptor directly. Definition of the cell membrane events that regulate factor V endocytosis will increase our understanding of how megakaryocytes acquire, process and package this critical coagulation factor, and may lead to the development of potential therapeutic targets in hypercoagulable and thrombotic states.